This is an application for a competing continuation of a long standing RO1 to study the chemistry and biochemical and clinical pharmacology of the antitumor agent cyclophosphamide (CP) and its close analog, ifosfamide (IF). CP was first introduced into clinical usage in the 1950's and remains one of the most widely used alkylating agents. It has a number of properties which contribute to its continued utility, including the fact that it exhibits relative sparing of the bone marrow and gi tract, as compared to other related agents. CP is remarkably immunosuppressive and is utilized in the treatment of a number of autoimmune diseases and is a major component of most bone marrow transplantation regimens. IF has similar pharmacologic properties and is preferentially used in the treatment of certain tumors. CP and IF are prodrugs which must be activated by hepatic P450. Competing with the activation reaction are secondary oxidations that lead to the production of neurotoxic metabolites. These undesirable oxidations are more prevalent with IF, making this drug a prime target for modification. In this renewal, we propose to continue and expand our studies of CP/IF alkylation, crosslinking and activation by determining those factors that are important in protecting against toxicities and critical to enhancing therapeutic effects. In Specific Aim 1, we will use various techniques including siRNA technology to determine the contributions of specific DNA-repair mechanisms to CP/IF resistance and toxicity. Identified mechanisms and genes could be targeted for inhibition with a result of relevant therapies for overcoming resistance. In Specific Aim 2 we will use human liver samples to evaluate the degree of human variation in the metabolism of CP and IF. We will also identify the SNPs in candidate genes including drug metabolizing genes or genes regulating these enzymes that are responsible for human variation in the phenotype. An understanding of the genotype(s) involved would allow for the identification of patients, before treatment, in whom the normal dose of CP or IF would be inadequate or toxic.